The goal of this project is to obtain a basic science information regarding therapeutic radiopharmaceuticals that are based on targeting the decay of Auger-electron-emitting radionuclides to specific sequences in genetic DNA. The principal innovation in our approach is that it is the specific DNA sequence of a gene within the genome of a cell that becomes the target of this "radiotherapy", not the total DNA of that cell. Gene-targetted "radiotherapy" optimally utilizes the sub-nanometer effect range of the Auger-electron-emitting radionuclides to allow targeting of most of the radiodamage to a selected gene sequence while producing minimal damage to the rest of the genome and to other cell components. &#8232;This approach requires a carrier molecule that exhibits enough specificity for a selected DNA sequence to deliver the radionuclide to that specific sequence and not to other sites in the genome. As our initial carrier molecule, we selected short synthetic oligonucleotides that are able to form a sequence-specific triple helix with the target DNA sequence, so-called triplex-forming oligonucleotides (TFO). We demonstrated the ability of 125I-TFO-NLS conjugates to produce double strand breaks in a specific site in the human multidrug resistance (mdr1) gene within cells. Currently we are developing a new generation of DNA sequence- and DNA structure-specific molecules, so-called peptide nucleic acids (PNA) consisting of DNA bases connected by peptide backbone. We designed PNA targeting a specific DNA structure (G-quadruplex) in the promoter region of the human BCL2 gene, and demonstrated their successful binding to the target sequence in vitro. We are working to optimize the delivery of short DNA and PNA molecules into the cell nucleus using a gamma-H2AX foci-formation assay detecting the damage to target DNA. We studied the mechanisms of DNA damage produced by decay of Auger-electron-emitting radionuclides. By using DNA fragments containing modified bases that served as barriers to electrons or holes migration we showed that charge migration along DNA contribute insignificantly to DNA damage after decay of 125I. Studying distributions of DNA breaks produced by decay of Auger emitters we found that frequencies of breaks strongly depend on the conformation of DNA molecule. This method, that we called radioprobing, was successfully applied to study DNA conformation within several DNA, RNA and DNA-protein structures. Currently. We are using radioprobing to determine conformation of G-quadruplex structures formed in the human telomeric sequences. We showed that conformation of these structures depends on the type of cations present in solution, i.e. Na or K, and that they could be highly polymorphic. We are completing studies using gene-expression analyses to examine the cellular responses to the DNA damage produced by Auger-decay effects in comparison with the gene expression patterns following external gamma irradiations of human embryonic stem cells. How IR affects the pluripotency of hESC is not understood. We found that irradiation of hESC of cultured H9 cell line with relatively low doses of 60Co gamma-radiation (0.2 Gy and 1 Gy) does not lead to loss of pluripotency capabilities of these cells. We also studied NIS expression during differentiation of hESC into thyroid-like cells; along with expression of other thyroid markers (TSHR, TPO, TG). Quantitative RT-PCR and immunostaining was used to study marker expression at all steps during differentiation, including pluripotency markers, germ layer markers and thyroid markers.